A Colorimetric LAMP Assay for Salmonella spp. Detection: Towards a DNA Extraction-Free Approach for Pathogen Screening.

Abstract

As of today, bacteriological identification and the molecular approach PCR are considered the gold standards for Salmonella spp. detection. However, these methods are time-consuming and costly due to the requirements for enrichment and nucleic acid extraction. In this study, we evaluated the reliability of a developed colorimetric loop-mediated isothermal amplification (cLAMP) assay targeting the hilA gene, using Phenol Red as an amplification indicator. Given that Phenol Red is pH-dependent, and to develop an extraction-free test, we evaluated chicken meat pretreatment and thermal treatment. First, we assessed the reliability of this test using a pure culture of Salmonella spp. and then in 50 chicken samples pretreated with optimal NaOH concentrations under standardized conditions. Samples representing extreme pH values were artificially contaminated and subjected to DNA extraction and a heat-treatment protocol. Serial dilutions of these products served as templates for LAMP reactions. The assay sensitivity was estimated to be around 3.9 CFU/µL of pure bacterial culture. In contrast, in biological samples, we detected up to 10 CFU/µL using DNA extraction, while heat treatment successfully amplified the initial solution and even some dilutions up to 10³ CFU/µL. In conclusion, our cLAMP assay demonstrated good sensitivity and provided clear evidence of its potential for in-field use without relying on prior enrichment steps and DNA extraction.